L-Thyroxine (T4) and bromoacetyl-L-thyroxine (BrAcT4) bind competitively to human serum prealbumin (PA). (14C)-Labeled BrAcT4 was, therefore, used to characterize the T4-binding site on PA by affinity labeling. Various degradation and fractionation methods permitted the determination of the points of attachment of the ligand in affinity-labeled PA. Gly-1, Lys-9, and Lys-15 had been labeled in a 30:61:9 ratio. Thyroxine-binding globulin (TBG) was purified from human plasma. Its amino acid composition was determined. Leu (1 mol/mol TBG) was found to be the carboxy- terminal amino acid. Thus, peptide mapping of a tryptic digest did not support a subunit structure recently claimed for TBG. Competitive binding of T4 and BrAcT4 to PA as well as binding of T4 to PA and to BrAcT4-treated PA indicate that the two ligands compete for the same binding site on PA.